Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, in many parts of the world, to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite $1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981; Seed Sci. & Technol. 9: 679-685).
The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981; Proc. 1981 Brit. Crop Prot. Conf.) contended that 24% of the powdery mildew populations from spring barley and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties, with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and Mycosphaerella fijiensis to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).
The three most important cereal crops in the world are maize (corn), rice and wheat (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 1). There are a great number of fungi, bacteria, and viruses that are pathogenic to maize, causing 9.4 % annual worldwide losses. In the corn belt of the United States, maize reduction because of disease infection is between 7 to 17% annually. Maize is the most important native American plant, and the U.S. produces about 44% of the world's 250 million metric tons annual production.
The major infectious diseases of maize are caused by fungi and include rusts, smuts, downy mildews, rots, spots, blights and deformations (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 13). Although fungal diseases are usually diagnosed by the structures produced by the pathogens, the differential symptomology caused by different isolates and species of these fungi make the accurate predictive determination of potential disease loss difficult. Consequently, the availability of improved diagnostic techniques for the rapid and accurate identification of specific pathogens will be of considerable use to field pathologists.
There are three primary species of Helminthosporium pathogenic to maize causing foliar diseases. Helminthosporium carbonum causes helminthosporium leaf spot (blight), also known as northern leaf spot (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 17). It is distributed throughout the Americas, southeast Asia, southeast Europe, south and central Africa, and India (Jones and Clifford; Cereal Diseases, John Wiley, 1983). There are two primary physiologically-based races. Race 1 is highly virulent on maize, causing a charred appearance on the ear's kernels. Race 2 tends to be less virulent than race 1 and does not diplay host specificity. Race 2 produces a host-specific toxin. Helminthosporium maydis causes southern leaf blight in maize. It occurs worldwide in warm (20-32.degree. C.), humid climates. In the United States, it is found in the southeastern and midwestern states (Jones and Clifford; Cereal Diseases, John Wiley, 1983). The disease was originally thought to be of little economic importance until a severe 1970 epidemic in the U.S. resulted in large losses. Northern leaf blight (turcicum leaf blight) is caused by Helminthosporium turcicum. The disease develops in humid areas of the world where maize is grown (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 16). Moderate temperatures (18-27.degree. C.) and heavy dews during the growing season promote severe disease development in which 50% losses of grain can occur. Typical control of these diseases include the use fungicides, crop rotation, burning crop debris, and breeding resistant hybrids and varieties.
Kabateilla zeae is another significant maize foliar pathogen causing eyespot disease. The disease originally reported as brown eyespot in Japan has also been found in Canada, Argentina, Austria, France, Germany, Yugoslavia, New Zealand and in several north-central U.S. states and Pennsylvania (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 21). The disease may develop on sheaths and outer husks, but lesions are more concentrated on leaves approaching maturity. In extremely infected plants, kernel infections may also develop. Cool, humid weather favors disease development. Disease control measures include the use of less susceptible hybrids, fungicides, and clean plowing or crop rotation.
Cercospora or gray leaf spot is caused by Cercospora zeae-maydisand infects maize, barnyardgrass, Johnsongrass and other Sorghum species (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 24). The disease is prevalent in warm-to-hot, humid areas of the United States, Mexico, Central America, northern South America, Europe, Africa, southeast Asia, India, China, and the Philippines. The disease has increased in severity in recent years in the southeastern and mid-Atlantic states of the U.S. especially in areas using minimum tillage of maize and no crop rotation (Latterell and Rossi, 1983; Plant Disease. Vol. 67, No. 8: 842-847). The disease can spread from the leaf sheaths to the stalk in highly infected plants. This can cause stalk deterioration to the point where lodging precludes mechanical harvesting. Crop rotation, resistant cultivars and fungicides are currently used to control gray leaf spot.
Puccinia sorghi causes common maize rust and can be found wherever maize is grown. Infection can occur on any plant parts above ground but is mainly found on the leaves (1973; Compendium of Corn Diseases, Amer. Phytopath. Soc. page 24). Cooler temperatures (16-23 .degree. C.) and high moisture contribute to the proliferation of the disease. Under severe infection conditions, chlorosis and death of the leaves and sheaths may occur ultimately reducing cereal yield.
Thus, there is a real need for the development of technology that will allow the identification of specific races of pathogen fungi early in the infection process. By identifying the specific race of a pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.
Additionally, with the increasing need for DNA fingerprinting, restriction fragment length polymorphism (RFLP) analysis, Southern transfers and PCR analysis, the isolation of high molecular weight DNA becomes a major problem when attempting to process a large number of plant samples in a timely manner. Several methods for the isolation of DNA have been reported, all of which have drawbacks for various reasons. These include DNA losses due to degradation and adsorption, the co-isolation of PCR inhibiting contaminants and labor extensive and costly protocols. Therefore, there is a need for a DNA extraction method which isolates high molecular weight DNA for high throughput analysis using molecular biology methods.